Revealing the Mechanisms and Physiology of Peroxisome Transfer in Stem Cell Therapy

项目摘要(摘要)。细胞间的通讯在所有组织和器官中都很重要，并通过 各种机制。细胞因子的分泌、缝隙连接蛋白、小分子和外切体 显示促进了细胞间的细胞质“信息”的交换。另一种新的蜂窝模式 通讯是细胞器转移的通讯，已被证明与线粒体一起发生。过氧酶体 是小的脂质结合的细胞器，负责极长链脂肪酸的代谢 手机。有几种罕见但毁灭性的儿童疾病是由过氧化物体缺陷引起的，如 肾上腺脑白质营养不良(ALD)。我们有新的证据表明，过氧化物体可以在不同的细胞类型之间转移。 我们假设，过氧化体转移可能是一种允许细胞稳定的机制 有功能失调的过氧化物酶。该应用程序的目标是开发一种动物模型，在该模型中 过氧化物酶体标记在造血细胞内，以允许过继转移实验和观察 过氧化物体在体外和体内的转运。目的1：优化斑马鱼过氧化体转移模型。我们的 在斑马鱼造血移植模型中产生的试点数据表明，捐赠者细胞可以 受体造血细胞的过氧化物体。如果我们能充分优化这一点，那将是最理想的 在HCT后的细胞剂量和时间方面。我们将确定过氧化酶体的最佳条件 在这个模型中，通过改变细胞剂量和移植读数后的时间来进行转移。转账将是 经流式细胞仪检测，经细胞分选和荧光显微镜验证。目的2：体外研究 不同细胞类型之间的过氧化体转移现象，并监测细胞功能的变化。我们 我创造了各种细胞系，并拥有含有过氧酶体标签序列的绿色荧光蛋白的原代细胞 (PTS-GFP)，允许特定的过氧酶体标记。我们将进行共培养实验来观察 并将量化将健康的过氧化物体转移到细胞中的生理效应 用功能失调的过氧酶体测量活性氧物种和线粒体功能。目标3： ROSA基因座控制下PTS-GFP表达小鼠的建立这一目标的目标是 PTS-GFP转基因小鼠群体的建立及其作为供体和受体的研究 实验使我们能够确定体内是否发生了过氧化物体转移。来自供体小鼠的HSPC将 移植到野生型小鼠体内。PTS-GFP小鼠也可以作为受体，以确定是否有 骨髓壁龛可以将过氧化物体转移到未标记的供体细胞中。这个项目既有高风险，又有 高额奖励，因为它的成功将开辟一个全新的领域，并将思维过程转化为过氧化物体、细胞器 传输和蜂窝通信。我们将获得新的知识并构建工具，不仅可以更好地理解 过氧化体转移，但开发新的策略、药物和小分子来增强这一过程 可能会影响我们如何治疗像肌萎缩侧索硬化症这样毁灭性的过氧酶体疾病。

Project Summary (Abstract). Cell-to-cell communication is important in all tissues and organs and occurs by a variety of mechanisms. Cytokine secretion, gap junction proteins, small molecules, and exosomes have been shown to facilitate the exchange of cytoplasmic “information” between cells. Another novel mode of cellular communication is that of organelle transfer which has been shown to occur with mitochondria. Peroxisomes are small lipid bound organelles which are responsible for the metabolism of very long chain fatty acids in the cell. There are several rare, but devastating childhood diseases that are a result of peroxisomal defects such adrenoleukodystrophy (ALD). We have new evidence that peroxisomes can be transferred between cell types. We hypothesize that peroxisomal transfer could be a mechanism that allows for the stabilization of cells harboring dysfunctional peroxisomes. The goal of this application is to develop an animal model in which peroxisomes are labeled within hematopoietic cells to allow adoptive transfer experiments and observation of peroxisomal transport in vitro and in vivo. Aim 1: Optimize the zebrafish model of peroxisomal transfer. Our pilot data, generated in a zebrafish model of hematopoietic transplant, indicate that donor cells can transfer peroxisomes to recipient hematopoietic cells. It would be ideal to understand if we can fully optimize this system in terms of cell dose and time after HCT. We will determine the optimal conditions for peroxisome transfer to occur in this model by varying the cell doses and time after transplant readout. Transfer will be determined by flow cytometry and verified by cell sorting and fluorescent microscopy. Aim 2: Study the in vitro phenomenon of peroxisomal transfer between various cell types and monitor changes in cellular function. We have created a variety of cell lines and have primary cells with GFP containing a peroxisome tag sequence (PTS-GFP) which allows for specific peroxisome labeling. We will perform co-culture experiments to observe peroxisomal transfer and will quantify the physiological effects of transferring healthy peroxisomes into cells with dysfunctional peroxisomes measuring reactive oxygen species and mitochondrial function. Aim 3: Generation of a PTS-GFP expressing mouse under the control of the Rosa locus. The goal of this aim is the establishment of a colony of PTS-GFP transgenic mice to be used as donors or recipients in adoptive experiments allowing us to determine if peroxisomal transfer occurs in vivo. HSPC from donor mice will transplanted into wild-type mice. PTS-GFP mice can also be used as recipients to determine if cells of the marrow niche can transfer peroxisomes into unlabeled donor cells. This project carries both high risk and high reward, as its success will open an entirely new field and thought process into peroxisomes, organelle transfer, and cellular communication. We will gain new knowledge and build tools to not only better understand peroxisomal transfer, but develop new strategies, drugs, and small molecules to enhance the process which could impact how we treated devastating peroxisomal disease like ALD.